Acalabrutinib as a novel hope for the treatment of breast and lung cancer: an in-silico proof of concept.

Drug repurposing is proved to be a groundbreaking concept in the field of cancer research, accelerating the pace of de novo drug discovery by investigating the anti-cancer activity of the already approved drugs. On the other hand, it got highly benefitted from the advancement in the in-silico tools and techniques, which are used to build up the initial "proof of concept" based on the drug-target interaction. Acalabrutinib (ACL) is a well-known drug for the treatment of hematological malignancies. But, the therapeutic ability of ACL against solid tumors is still unexplored. Thereby, the activity of ACL on breast cancer and lung cancer was evaluated utilizing different computational methods. A series of proteins such as VEGFR1, ALK, BCL2, CXCR-4, mTOR, AKT, PI3K, HER-2, and Estrogen receptors were selected based on their involvement in the progression of the breast as well as lung cancer. A multi-level computational study starting from protein-ligand docking to molecular dynamic (MD) simulations were performed to detect the binding potential of ACL towards the selected proteins. Results of the study led to the identification of ACL as a ligand that showed a high docking score and binding energy with HER-2, mTOR, and VEGFR-1 successively. Whereas, the MD simulations study has also shown good docked complex stability of ACL with HER2 and VEGFR1. Our findings suggest that interaction with those receptors can lead to preventive action on both breast and lung cancer, thus it can be concluded that ACL could be a potential molecule for the same purpose.Communicated by Ramaswamy H. Sarma.

Single Amino Acid Polymorphisms in the Fasciola hepatica Carboxylesterase Type B Gene and Their Potential Role in Anthelmintic Resistance.

The expression of the Fasciola hepatica carboxylesterase type B (CestB) gene is known to be induced upon exposure to the anthelmintic triclabendazole (TCBZ), leading to a substantial rise in enzyme-specific activity. Furthermore, the nucleotide sequence of the CestB gene displays variations that can potentially result in radical amino acid substitutions at the ligand binding site. These substitutions hold the potential to impact both the ligand-protein interaction and the catalytic properties of the enzyme. Thus, the objective of our study was to identify novel CestB polymorphisms in TCBZ-resistant parasites and field isolates obtained from a highly endemic region in Central Mexico. Additionally, we aimed to assess these amino acid polymorphisms using 3D modeling against the metabolically oxidized form of the anthelmintic TCBZSOX. Our goal was to observe the formation of TCBZSOX-specific binding pockets that might provide insights into the role of CestB in the mechanism of anthelmintic resistance. We identified polymorphisms in TCBZ-resistant parasites that exhibited three radical amino acid substitutions at positions 147, 215, and 263. These substitutions resulted in the formation of a TCBZSOX-affinity pocket with the potential to bind the anthelmintic drug. Furthermore, our 3D modeling analysis revealed that these amino acid substitutions also influenced the configuration of the CestB catalytic site, leading to alterations in the enzyme's interaction with chromogenic carboxylic ester substrates and potentially affecting its catalytic properties. However, it is important to note that the TCBZSOX-binding pocket, while significant for drug binding, was located separate from the enzyme's catalytic site, rendering enzymatic hydrolysis of TCBZSOX impossible. Nonetheless, the observed increased affinity for the anthelmintic may provide an explanation for a drug sequestration type of anthelmintic resistance. These findings lay the groundwork for the future development of a molecular diagnostic tool to identify anthelmintic resistance in F. hepatica.

Investigation of Potential Drug Targets for Cholesterol Regulation to Treat Alzheimer's Disease.

Despite extensive research and seven approved drugs, the complex interplay of genes, proteins, and pathways in Alzheimer's disease remains a challenge. This implies the intricacies of the mechanism for Alzheimer's disease, which involves the interaction of hundreds of genes, proteins, and pathways. While the major hallmarks of Alzheimer's disease are the accumulation of amyloid plaques and tau protein tangles, excessive accumulation of cholesterol is reportedly correlated with Alzheimer's disease patients. In this work, protein-protein interaction analysis was conducted based upon the genes from a clinical database to identify the top protein targets with most data-indicated involvement in Alzheimer's disease, which include ABCA1, CYP46A1, BACE1, TREM2, GSK3B, and SREBP2. The reactions and pathways associated with these genes were thoroughly studied for their roles in regulating brain cholesterol biosynthesis, amyloid beta accumulation, and tau protein tangle formation. Existing clinical trials for each protein target were also investigated. The research indicated that the inhibition of SREBP2, BACE1, or GSK3B is beneficial to reduce cholesterol and amyloid beta accumulation, while the activation of ABCA1, CYP46A1, or TREM2 has similar effects. In this study, Sterol Regulatory Element-Binding Protein 2 (SREBP2) emerged as the primary protein target. SREBP2 serves a pivotal role in maintaining cholesterol balance, acting as a transcription factor that controls the expression of several enzymes pivotal for cholesterol biosynthesis. Novel studies suggest that SREBP2 performs a multifaceted role in Alzheimer's disease. The hyperactivity of SREBP2 may lead to heightened cholesterol biosynthesis, which suggested association with the pathogenesis of Alzheimer's disease. Lowering SREBP2 levels in an Alzheimer's disease mouse model results in reduced production of amyloid-beta, a major contributor to Alzheimer's disease progression. Moreover, its thoroughly analyzed crystal structure allows for computer-aided screening of potential inhibitors; SREBP2 is thus selected as a prospective drug target. While more protein targets can be added onto the list in the future, this work provides an overview of key proteins involved in the regulation of brain cholesterol biosynthesis that may be further investigated for Alzheimer's disease intervention.

Binding Affinity and Mechanisms of Potential Antidepressants Targeting Human NMDA Receptors.

Depression, a mental disorder that plagues the world, is a burden on many families. There is a great need for new, fast-acting antidepressants to be developed. N-methyl-D-aspartic acid (NMDA) is an ionotropic glutamate receptor that plays an important role in learning and memory processes and its TMD region is considered as a potential target to treat depression. However, due to the unclear binding sites and pathways, the mechanism of drug binding lacks basic explanation, which brings great complexity to the development of new drugs. In this study, we investigated the binding affinity and mechanisms of an FDA-approved antidepressant (S-ketamine) and seven potential antidepressants (R-ketamine, memantine, lanicemine, dextromethorphan, Ro 25-6981, ifenprodil, and traxoprodil) targeting the NMDA receptor by ligand-protein docking and molecular dynamics simulations. The results indicated that Ro 25-6981 has the strongest binding affinity to the TMD region of the NMDA receptor among the eight selected drugs, suggesting its potential effective inhibitory effect. We also calculated the critical binding-site residues at the active site and found that residues Leu124 and Met63 contributed the most to the binding energy by decomposing the free energy contributions on a per-residue basis. We further compared S-ketamine and its chiral molecule, R-ketamine, and found that R-ketamine had a stronger binding capacity to the NMDA receptor. This study provides a computational reference for the treatment of depression targeting NMDA receptors, and the proposed results will provide potential strategies for further antidepressant development and is a useful resource for the future discovery of fast-acting antidepressant candidates.

Improved docking of peptides and small molecules in iMOLSDOCK.

iMOLSDOCK is an induced-fit docking algorithm that uses the mutually orthogonal Latin squares (MOLS) sampling technique. Here, we describe the updates made to iMOLSDOCK in order to increase receptor flexibility, improve the scoring system, and speed up calculation. With a dataset of 35 peptide-protein complexes, the PepSet benchmark dataset of 80 peptide-protein complexes, and the Astex Diverse set, which uses nonpeptide small molecules as ligands, iMOLSDOCK has been benchmarked and validated. Flexible residues are now able to deviate from the starting position by a maximum of 3.0 Å due to the increased receptor flexibility. The ranking effectiveness of iMOLSDOCK has increased by 24% once the scoring system was improved. Additionally, iMOLSDOCK has been compared to Gold v5.2.1, HPEPDOCK, AutoDock CrankPep v1.0, AutoDock Vina, HADDOCK, PatchDock, and RosettaLigand. For induced-fit peptide-protein docking, iMOLSDOCK achieved success rates of 6%, 37%, and 89% at the top 1, 10, and 100 levels. At the top 1, 10, and 100 levels, iMOLSDOCK had success rates for small molecule-protein docking of 14%, 31%, and 49%. The computation time for peptide docking was lowered by two orders of magnitude, and for nonpeptide small molecule docking, it was roughly 14 times faster due to code optimization in the iMOLSDOCK docking tool. Source code and binary of iMOLSDOCK could be obtained from https://sourceforge.net/projects/mols2-0/files/ .

Lysophosphatidic acid signaling via LPA6 : A negative modulator of developmental oligodendrocyte maturation.

The developmental process of central nervous system (CNS) myelin sheath formation is characterized by well-coordinated cellular activities ultimately ensuring rapid and synchronized neural communication. During this process, myelinating CNS cells, namely oligodendrocytes (OLGs), undergo distinct steps of differentiation, whereby the progression of earlier maturation stages of OLGs represents a critical step toward the timely establishment of myelinated axonal circuits. Given the complexity of functional integration, it is not surprising that OLG maturation is controlled by a yet fully to be defined set of both negative and positive modulators. In this context, we provide here first evidence for a role of lysophosphatidic acid (LPA) signaling via the G protein-coupled receptor LPA6 as a negative modulatory regulator of myelination-associated gene expression in OLGs. More specifically, the cell surface accessibility of LPA6 was found to be restricted to the earlier maturation stages of differentiating OLGs, and OLG maturation was found to occur precociously in Lpar6 knockout mice. To further substantiate these findings, a novel small molecule ligand with selectivity for preferentially LPA6 and LPA6 agonist characteristics was functionally characterized in vitro in primary cultures of rat OLGs and in vivo in the developing zebrafish. Utilizing this approach, a negative modulatory role of LPA6 signaling in OLG maturation could be corroborated. During development, such a functional role of LPA6 signaling likely serves to ensure timely coordination of circuit formation and myelination. Under pathological conditions as seen in the major human demyelinating disease multiple sclerosis (MS), however, persistent LPA6 expression and signaling in OLGs can be seen as an inhibitor of myelin repair. Thus, it is of interest that LPA6 protein levels appear elevated in MS brain samples, thereby suggesting that LPA6 signaling may represent a potential new druggable pathway suitable to promote myelin repair in MS.

Sarcorucinine-D Inhibits Cholinesterases and Calcium Channels: Molecular Dynamics Simulation and In Vitro Mechanistic Investigations.

Acetylcholinesterase (AChE) inhibitors and calcium channel blockers are considered effective therapies for Alzheimer's disease. AChE plays an essential role in the nervous system by catalyzing the hydrolysis of the neurotransmitter acetylcholine. In this study, the inhibition of the enzyme AChE by Sarcorucinine-D, a pregnane type steroidal alkaloid, was investigated with experimental enzyme kinetics and molecular dynamics (MD) simulation techniques. Kinetics studies showed that Sarcorucinine-D inhibits two cholinesterases-AChE and butyrylcholinesterase (BChE)-noncompetitively, with Ki values of 103.3 and 4.66 µM, respectively. In silico ligand-protein docking and MD simulation studies conducted on AChE predicted that Sarcorucinine-D interacted via hydrophobic interactions and hydrogen bonds with the residues of the active-site gorge of AChE. Sarcorucinine-D was able to relax contractility concentration-dependently in the intestinal smooth muscles of jejunum obtained from rabbits. Not only was the spontaneous spasmogenicity inhibited, but it also suppressed K+-mediated spasmogenicity, indicating an effect via the inhibition of voltage-dependent Ca2+ channels. Sarcorucinine-D could be considered a potential lead molecule based on its properties as a noncompetitive AChE inhibitor and a Ca2+ channel blocker.

The Anti-Tumor Mechanism and Target of Triptolide Based on Network Pharmacology and Molecular Docking.

BACKGROUND: According to the special physiological and pharmacological activities of natural compounds, many drugs with special therapeutic effects have been developed. The Triptolide (TP) is a natural anti-tumor drug with a world patent, but its target and mechanism are yet unknown. OBJECTIVE: The study aims to explore and predict the target and mechanism of TP on Non-Small Cell Lung Cancer (NSCLC), Pancreatic Cancer (PC) and Colorectal Cancer (CC) through network pharmacology technology. METHODS: We screened the core targets of TP with NSCLC, PC and CC, respectively, and carried out network analysis, enrichment analysis and ligand-receptor docking to clarify its potential pharmacological mechanism. RESULTS: By screening the core genes between TP with NSCLC, PC and CC, respectively, it was found that PTGS2 was the common target gene in the three cancers. NSCLC, CCL2, IL6, HMOX1 and COL1A1 are the specific target genes, while MMP2, JUN, and CXCL8 are the specific target genes in PC. In CC, the specific target genes includeERBB2, VEGFA, STAT1 and MAPK8. In enrichment analysis, it was found that the NF- κB, toll-like receptors and IL-17 signaling pathway were mainly involved in TP for these cancers. The binding energy of TP to the core target is less than that of cyclophosphamide. CONCLUSION: This study preliminarily revealed that TP may prevent and treat cancers\ through multiple targets and pathways. The possible mechanisms of TP include regulating immune and inflammatory responses, promoting apoptosis and inhibiting tumor development. It shows that TP may have potential in treating kinds of tumors.

Potential inhibitors for the novel coronavirus (SARS-CoV-2).

The lack of a vaccine or any effective treatment for the aggressive novel coronavirus disease (COVID-19) has created a sense of urgency for the discovery of effective drugs. Several repurposing pharmaceutical candidates have been reported or envisaged to inhibit the emerging infections of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but their binding sites, binding affinities and inhibitory mechanisms are still unavailable. In this study, we use the ligand-protein docking program and molecular dynamic simulation to ab initio investigate the binding mechanism and inhibitory ability of seven clinically approved drugs (Chloroquine, Hydroxychloroquine, Remdesivir, Ritonavir, Beclabuvir, Indinavir and Favipiravir) and a recently designed α-ketoamide inhibitor (13b) at the molecular level. The results suggest that Chloroquine has the strongest binding affinity with 3CL hydrolase (Mpro) among clinically approved drugs, indicating its effective inhibitory ability for SARS-CoV-2. However, the newly designed inhibitor 13b shows potentially improved inhibition efficiency with larger binding energy compared with Chloroquine. We further calculate the important binding site residues at the active site and demonstrate that the MET 165 and HIE 163 contribute the most for 13b, while the MET 165 and GLN 189 for Chloroquine, based on residual energy decomposition analysis. The proposed work offers a higher research priority for 13b to treat the infection of SARS-CoV-2 and provides theoretical basis for further design of effective drug molecules with stronger inhibition.

In silico identification of drug candidates against COVID-19.

The COVID-19 pandemic has caused unprecedented health and economic crisis throughout the world. However, there is no effective medication or therapeutic strategy for treatment of this disease currently. Here, to elucidate the inhibitory effects, we first tested binding affinities of 11 HIV-1 protease inhibitors or their pharmacoenhancers docked onto SARS-CoV-2 main protease (M pro ), and 12 nucleotide-analog inhibitors docked onto RNA dependent RNA polymerase (RdRp). To further obtain the effective drug candidates, we screened 728 approved drugs via virtual screening on SARS-CoV-2 M pro . Our results demonstrate that remdesivir shows the best binding energy on RdRp and saquinvir is the best inhibitor of M pro . Based on the binding energies, we also list 10 top-ranked approved drugs which can be potential inhibitors for M pro . Overall, our results do not only propose drug candidates for further experiments and clinical trials but also pave the way for future lead optimization and drug design.

A Pilot Study of All-Computational Drug Design Protocol-From Structure Prediction to Interaction Analysis.

Speeding up the drug discovery process is of great significance. To achieve that, high-efficiency methods should be exploited. The conventional wet-bench methods hardly meet the high-speed demand due to time-consuming experiments. Conversely, in silico approaches are much more efficient for drug discovery and design. However, in silico approaches usually serve as a supportive role in research processes. To fully exert the strength of computational methods, we propose a protocol which integrates various in silico approaches, from de novo protein structure prediction to ligand-protein interaction simulation. As a proof of concept, human SK2/calmodulin complex was used as a target for validation. First, we obtained a predicted structure of SK2/calmodulin and predicted binding sites which were consistent with the literature data. Then we investigated the ligand-protein interaction via virtual mutagenesis, flexible docking, and binding affinity calculation. As a result, the binding energies of mutants have similar trends compared with the EC50 values (R = 0.6 for NS309 in V481 mutants). The results indicate that our protocol can be applied to the drug design of structure unknown proteins. Our study also demonstrates that the integration of in silico approaches is feasible and it facilitates the acceleration of new drug discovery.

An integrated strategy for identifying new targets and inferring the mechanism of action: taking rhein as an example.

BACKGROUND: Target identification is necessary for the comprehensive inference of the mechanism of action of a compound. The application of computational methods to predict the targets of bioactive compounds saves cost and time in drug research and development. Therefore, we designed an integrated strategy consisting of ligand-protein docking, network analysis, enrichment analysis, and an experimental surface plasmon resonance (SPR) method to identify and validate new targets, and then used enriched pathways to elucidate the underlying pharmacological mechanisms. Here, we used rhein, a compound with various pharmacological activities, as an example to find some of its previously unknown targets and to determine its pharmacological activity. RESULTS: A total of nine candidate targets were discovered, including LCK, HSP90AA1, RAB5A, EGFR, CDK2, CDK6, GSK3B, p38, and JNK. LCK was confirmed through SPR experiments, and HSP90AA1, EGFR, CDK6, p38, and JNK were validated through previous reports. Rhein network regulations are complex and interconnected. The therapeutic effect of rhein is the synergistic and comprehensive result of this vast and complex network, and the perturbation of multiple targets gives rhein its various pharmacological activities. CONCLUSIONS: This study provided a new integrated strategy to identify new targets of bioactive compounds and reveal their molecular mechanisms of action.

A molecular docking study of the interactions between human transferrin and seven metallocene dichlorides.

Human Transferrin (hTf) is a metal-binding protein found in blood plasma and is well known for its role in iron delivery. With only a 30% of its capacity for Fe+3 binding, this protein has the potential ability to transport other metal ions or organometallic compounds from the blood stream to all cell tissues. In this perspective, recent studies have described seven metallocene dichlorides (Cp2M(IV)Cl2, M(IV)=V, Mo, W, Nb, Ti, Zr, Hf) suitable as anticancer drugs and less secondary effects than cisplatin. However, these studies have not provided enough data to clearly explain how hTf binds and transports these organometallic compounds into the cells. Thus, a computational docking study with native apo-hTf using Sybyl-X 2.0 program was conducted to explore the binding modes of these seven Cp2M(IV)Cl2 after their optimization and minimization using Gaussian 09. Our model showed that the first three Cp2M(IV)Cl2 (M(IV)=V, Mo, W) can interact with apo-hTf on a common binding site with the amino acid residues Leu-46, Ile-49, Arg-50, Leu-66, Asp-69, Ala-70, Leu-72, Ala-73, Pro-74 and Asn-75, while the next four Cp2M(IV)Cl2 (M(IV)=Nb, Ti, Zr, Hf) showed different binding sites, unknown until now. A decreasing order in the total score (equal to -log Kd) was observed from these docking studies: W (5.4356), Mo (5.2692), Nb (5.1672), V (4.5973), Ti (3.6529), Zr (2.0054) and Hf (1.8811). High and significant correlation between the affinity of these seven ligands (metallocenes) for apo-hTf and their bond angles CpMCp (r=0.94, p<0.01) and Cl-M-Cl (r=0.95, p<0.01) were observed, thus indicating the important role that these bond angles can play in ligand-protein interactions. Fluorescence spectra of apo-hTf, measured at pH 7.4, had a decrease in the fluorescence emission spectrum with increasing concentration of Cp2M(IV)Cl2. Experimental data has a good correlation between KA (r=0.84, p=0.027) and Kd (r=0.94, p=0.0014) values and the calculated total scores obtained from our docking experiments. In conclusion, these results suggest that the seven Cp2M(IV)Cl2 used for this study can interact with apo-hTf, and their affinity was directly and inversely proportional to their bond angles CpMCp and ClMCl, respectively. Our docking studies also suggest that the binding of the first three Cp2M(IV)Cl2 (M(IV)=V, Mo, W) to hTf could abrogate the formation of the hTf-receptor complex, and as a consequence the metallocene-hTf complex might require another transport mechanism in order to get into the cell.

Binding of the anticancer drug BI-2536 to human serum albumin. A spectroscopic and theoretical study.

BI-2536 is a potent Polo-like kinase inhibitor which induces apoptosis in diverse human cancer cell lines. The binding affinity of BI-2536 for human serum albumin (HSA) protein may define its pharmacokinetic and pharmacodynamic profile. We have studied the binding of BI-2536 to HSA by means of different spectroscopic techniques and docking calculations. We have experimentally observed that the affinity of BI-2536 for HSA is higher than that of other common HSA binding drugs. Therefore, it can be postulated that the drug dose should be increased to achieve a certain concentration of free drug in plasma, although BI-2536 could also reach tumour tissues by uptaking HSA/BI-2536 complex. Only a single binding site on HSA has been observed for BI-2536 which seems to correspond to the subdomain IIA pocket. The formation of the HSA/BI-2536 complex is a spontaneous and entropy-driven process that does not cause a significant change of the secondary structure of the protein. Its endothermic character could be related to proton release. Thermodynamic analysis showed that the main protein-drug interactions are of the van der Waals type although the presence of amide and ether groups in BI-2536 could also allow H-bonding with some residues in the subdomain IIA pocket.

Computational study of pH-dependent oligomerization and ligand binding in Alt a 1, a highly allergenic protein with a unique fold.

Alt a 1 is a highly allergenic protein from Alternaria fungi responsible for several respiratory diseases. Its crystal structure revealed a unique ß-barrel fold that defines a new family exclusive to fungi and forms a symmetrical dimer in a butterfly-like shape as well as tetramers. Its biological function is as yet unknown but its localization in cell wall of Alternaria spores and its interactions in the onset of allergy reactions point to a function to transport ligands. However, at odds with binding features in ß-barrel proteins, monomeric Alt a 1 seems unable to harbor ligands because the barrel is too narrow. Tetrameric Alt a 1 is able to bind the flavonoid quercetin, yet the stability of the aggregate and the own ligand binding are pH-dependent. At pH 6.5, which Alt a 1 would meet when secreted by spores in bronchial epithelium, tetramer-quercetin complex is stable. At pH 5.5, which Alt a 1 would meet in apoplast when infecting plants, the complex breaks down. By means of a combined computational study that includes docking calculations, empirical pKa estimates, Poisson-Boltzmann electrostatic potentials, and Molecular Dynamics simulations, we identified a putative binding site at the dimeric interface between subunits in tetramer. We propose an explanation on the pH-dependence of both oligomerization states and protein-ligand affinity of Alt a 1 in terms of electrostatic variations associated to distinct protonation states at different pHs. The uniqueness of this singular protein can thus be tracked in the combination of all these features.

Synthesis and antibacterial activity of some novel 4-oxopyrido[2,3-a]phenothiazines.

A series of substituted 4-oxopyrido[2,3-a]phenothiazine-3-carboxylic acids (6a-d) were prepared via cyclization of the corresponding ethyl 7-(arylthioxy)-8-nitro(or azido)-4-oxoquinoline-3-carboxylates (3a-d/4a-d), followed by hydrolysis of the resultant esters (5a-d). Among these tetracyclics, compound 6a with unsubstituted terminal benzo-ring D was the most active against representative Gram-positive and Gram-negative bacterial strains. These compounds were also active against methicillin-resistant Staphylococcus aureus (MRSA), with very low toxicity to normal cells. Virtual screening using ligand-protein docking modeling predicted that the compounds 6a-d are potential inhibitors of the topoisomerase IV enzyme and that hydrophobic interactions and hydrogen bonds are the major molecular interactions between these compounds and the residues of the active site of topoisomerase IV.

Utilization of 6-(methylsulfinyl)hexyl isothiocyanate for sensitization of tumor cells to antitumor agents in combination therapies.

In the present study, we performed in silico and in vitro analyses to evaluate the chemosensitizing effects of 6-(methylsulfinyl)hexyl isothiocyanate (6-MITC) on tumor cells. Our in silico analyses of the ligand-receptor interactions between 6-MITC and the glutamate cysteine ligase (GCL) catalytic subunit (GCLC) revealed that 6-MITC possibly inhibited GCL enzyme activity, and that Cys-249 and Gln-251 were important residues for stable binding of ligands to GCLC. It was further found that 6-MITC interfered with the hydrogen bonds of the cysteinyl and glutamyl moieties of GSH with Cys-249 and Gln-251, respectively, and possibly overrode the feedback inhibition of GCL enzyme activity by GSH. To the best of our knowledge, this is the first in silico analysis to suggest an overriding effect of 6-MITC on GSH-induced feedback inhibition of GCL. In our in vitro analyses, combined treatment with 6-MITC and L-buthionine-S,R-sulfoximine (BSO) depleted GSH within 4 h in tumorigenic human c-Ha-ras and mouse c-myc-cotransfected highly metastatic serum-free mouse embryo-1 (r/m HM-SFME-1) cells, but did not deplete GSH in normal SFME cells. Furthermore, exposure to 6-MITC plus BSO for 4h, followed by glycyrrhetinic acid (GA) treatment for 3h, eradicated the tumor cells with minimal damage to the normal cells. The present findings suggest that 6-MITC in combination therapies could be used to sensitize tumor cells to antitumor agents, thereby leading to their eradication.